Thermacetogenium phaeum consortium for the production of materials with enhanced hydrogen content

ABSTRACT

An isolated microbial consortium is described that includes a first microbial consortium having  Thermacetogenium phaeum  to metabolize a complex hydrocarbon substrate into metabolic products comprising an acetate compound. The consortium also includes a second microbial consortium having a methanogen to convert the acetate compound into a final product that includes methane. Also, a method of increasing production of materials with enhanced hydrogen content. The method includes isolating  Thermacetogenium phaeum  from geologic formation water, culturing the isolated  Thermacetogenium phaeum  to increase the  Thermacetogenium phaeum  population, and introducing a consortium of the cultured  Thermacetogenium phaeum , which may include spores of  Thermacetogenium phaeum , into a hydrocarbon formation containing a complex hydrocarbon substrate.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/330,759, filed Jan. 11, 2006, entitled “THERMACETOGENIUM PHAEUMCONSORTIUM FOR THE PRODUCTION OF MATERIALS WITH ENHANCED HYDROGENCONTENT,” the entire disclosure of which is incorporated herein byreference.

FIELD OF THE INVENTION

The invention relates generally to the development of microbialconsortium of microorganisms that can be used in formation environmentsto enhance metabolism of complex organic substrates (e.g. oil) intosimpler compounds, such as methane. The invention also relates tomethods of isolating microorganisms such as Thermacetogenium phaeum fromtheir native environments.

BACKGROUND OF THE INVENTION

The United States and the rest of world are increasingly relying onnatural gas for heating homes and buildings, generating electric powerfor residential and industrial applications, and as a feedstock for avariety of synthetic organic chemicals. Future demand for natural gascould grow even more dramatically as more new cars and trucks useelectric power. Natural gas will likely play a major role in generatingthe electricity used in charging vehicle batteries, and supplying themethane and hydrogen used in operating vehicle fuel cells. Because thealready high demand for natural gas is expected to soar, concerns aboutsupply are growing.

One proposed solution is to build more long haul infrastructure to movenatural gas where demand is greatest. Because natural gas is a gas atroom temperature, there are more challenges in transporting it than forfuels like oil, coal and gasoline. The current leading proposal is tocool the gas to temperatures where it liquefies and transport the liquidnatural gas (“LNG”) on refrigerated tankers. At the destination port,the LNG is warmed back into a gas that gets fed into the existing gaspipeline infrastructure. This proposed solution has been criticized fora number or reasons, including the significant cost associated withcondensing and transporting LNG. There are also serious safety concernsabout accidents and sabotage to the LNG infrastructure. These concernshave lead to strong local opposition to proposed sites for building aLNG port. In addition, some have argued that importing LNG onlyincreases dependence on foreign sources of natural gas from politicallyunstable regions.

Another proposed solution is to explore and develop more sites for localnatural gas production. In the United States, one site believed tocontain large, untapped reserves of natural gas is the Arctic NationalWildlife Refuge (“ANWR”) in Alaska. But proposals to develop this site,as well as other sites in the Rocky Mountain region of the UnitedStates, have been strongly opposed by environmental groups. Thus,development of new sources of local natural gas also faces significantlimitations.

One way to avoid the problems with importing natural gas and developingnew sites is to enhance gas production from existing local sites. Onepromising technology for doing this is microbially enhanced methaneproduction. This technology uses microorganisms to metabolize complexorganic substrates such as oil or coal into simpler compounds likemethane and hydrogen. Stimulating the right indigenous microorganisms orintroducing the right microorganisms under the right conditions to“retired” oil fields and coal deposits that are too deep to mine couldturn these sites into productive new sources of domestic natural gas.

Because only a small fraction of the oil and coal was recovered fromthese sites while they were operating, the amount of hydrocarbonsubstrate still available for microbial conversion is enormous. Themature coal mines in the Powder River Basin in Wyoming, for example, arestill estimated to have 1,300 billion short tons of unmined coal. Ifjust 1% of this coal could be microbially converted into natural gas, itcould supply the current annual natural gas need of the United Statesfor four years. This story could be repeated at many other mature coaland oil sites across the country.

But the technology of microbial natural gas generation is still in itsinfancy. Many of the microorganisms require an anoxic or microoxicenvironment and are difficult to study in a conventional microbiologylaboratory. While metabolic pathways have been postulated for breakingdown a complex hydrocarbon substrate into natural gas and hydrogen, fewspecific microorganisms that are involved in methanogenic hydrocarbonbiodegradation have been positively identified. The “first bite”microorganisms, which start the initial breakdown of complex hydrocarbonsubstrate into smaller molecules, have been particularly elusive.

Thus, there is a need for identifying microorganisms involved inbiogenic gas production in underground hydrocarbon formations such ascoal mines and oil fields. The identity of these microorganisms willprovide valuable insight into how complex hydrocarbons are convertedinto fuel gases like hydrogen and methane. This insight can lead tomethods of isolating, cultivating, and stimulating the microorganisms tomake fuel gases at commercially viable production rates. These and otherissues are addressed by the present invention.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention include isolated microbial consortium. Theconsortium include a first microbial consortium that hasThermacetogenium phaeum to metabolize a complex hydrocarbon substrateinto metabolic products comprising an acetate compound. The consortiummay also include a second microbial consortium that has a methanogen toconvert the acetate compound into a final product comprising methane.

Embodiments of the invention may also include methods of increasingproduction of materials with enhanced hydrogen content. The methods mayinclude the steps of isolating Thermacetogenium phaeum from geologicformation water, and culturing the isolated Thermacetogenium phaeum toincrease the Thermacetogenium phaeum population. The methods may furtherinclude introducing a consortium comprising the culturedThermacetogenium phaeum into a hydrocarbon formation containing acomplex hydrocarbon substrate.

Embodiments of the invention still further include methods of isolatingThermacetogenium phaeum from a native consortium of microorganisms. Themethods may include the steps of removing formation water from anunderground formation, where the formation water includes the nativeconsortium that includes the Thermacetogenium phaeum. The methods mayalso include heating the formation water to about 120° C. or more, wherethe Thermacetogenium phaeum, including its spores, survives the heatingprocess. The heating may cause at least some of the Thermacetogeniumphaeum to form spores (i.e., to sporulate).

Embodiments of the invention still also include methods of stimulatingbiological activity of Thermacetogenium phaeum in a geologic formation.The methods may include the steps of detecting the Thermacetogeniumphaeum in the geologic formation, measuring one or more environmentalcharacteristics of the geologic formation, and determining a compositionof carbonaceous material in the formation. The methods may also includeadding an amendment to the formation based on the measurements of theenvironmental characteristics and the composition of the carbonaceousmaterial.

Additional embodiments and features are set forth in part in thedescription that follows, and in part will become apparent to thoseskilled in the art upon examination of the specification or may belearned by the practice of the invention. The features and advantages ofthe invention may be realized and attained by means of theinstrumentalities, combinations, and methods described in thespecification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a simplified schematic of a three-stage anaerobicconversion of a complex hydrocarbon substrate to a material with anenhanced hydrogen content;

FIG. 2A is a flowchart illustrating steps in methods of isolating aconsortium of microorganisms and preparing a consortium that includesmicroorganisms according to embodiments of the invention. FIG. 2B is aflowchart illustrating steps in methods of isolating a consortium ofmicroorganisms and preparing a consortium that includes microorganismsaccording to embodiments of the invention;

FIG. 3 is a flowchart of steps in methods of stimulating biologicalactivity of Thermoacetogenium Phaeum according to embodiments of theinvention;

FIG. 4A is a graph of the accumulation of acetate in a formation watersample. FIG. 4B is a graph of the accumulation of methane in a formationwater sample;

FIG. 5 is a TRFLP banding pattern of DNA samples taken from consortiummicroorganisms; and

FIG. 6A is a graph of acetate concentrations in autoclaved formationwater. FIG. 6B is a graph of acetate concentrations in unamendedincubations.

DETAILED DESCRIPTION OF THE INVENTION

Thermacetogenium phaeum (“T. phaeum”) is identified as a “first bite”microorganism that can metabolize part of a complex hydrocarbonsubstrate such as crude oil into a fermentation end product such asacetate (e.g., acetic acid) or formate (e.g., formic acid). Thus, T.phaeum appears to play dual roles in a metabolic process of convertingcomplex hydrocarbons to methane: The microorganism appears to beresponsible for the hydrolytic fermentation of the starting hydrocarbonto an intermediate, and also for converting that intermediate intoacetate. After this, all that remains is to convert the acetate intomethane with a methanogen. The acetate can be converted directly tomethane. Alternatively, a syntrophic microorganism can convert theacetate to hydrogen gas and carbon dioxide, which is utilized byhydrogen utilizing methanogens. Thus, a process for metabolizing acomplex hydrocarbon into methane has been discovered that requires onlytwo or three species of anaerobic microorganisms.

The discovery of a two member consortium for biogenic gas productionmeans that processes of stimulating biogenic gas production may besimpler than previously thought. Metabolic processes that requireparticipation from several species of microorganisms increase thechances for a bottleneck when the metabolism of one species starts todecline. In addition, environmental changes, such as a change intemperature, nutrient availability, inhibitor accumulation, pH, etc.,that stimulate the participation of one species in the metabolic processmay hurt participation by others. When fewer species are used, there'san increased chance for the successful and prolonged stimulation ofmethane or hydrogen formation in subsurface formations.

Embodiments of the invention include a consortium of isolatedmicroorganisms that may require two species to metabolize a complexhydrocarbon substrate like oil into biogenic gases like methane orhydrogen. One member of this consortium is T. phaeum and a second membermay be a methanogen such as a Methanobacteriales, Methanomircobacteria,Methanopyrales, Methanococcales, Methanosaeta, etc. The consortium mayalso include additional microorganism species that provide additionalmetabolic pathways to the final products. For example, the consortiummay include multiple species of methanogens: One species can convert theacetate produced by T. phaeum directly into methane and carbon dioxidethough an acetate fermentation process. Another species can combine thecarbon dioxide with hydrogen to produce methane and water via acarbonate reduction. Additional details on the role of T. phaeum inmetabolic processes to convert a complex hydrocarbon substrate tomaterials with an enhanced hydrogen content will now be described.

Biogenic Processes of Making Materials with Enhanced Hydrogen Content

FIG. 1 shows a simplified schematic of a three-stage anaerobicconversion of a complex hydrocarbon substrate to a material with anenhanced hydrogen content. Additional details about three stagemetabolic processes may be found in co-assigned U.S. patent applicationSer. No. 11/099,881, titled “GENERATION OF MATERIALS WITH ENHANCEDHYDROGEN CONTENT FROM ANAEROBIC MICROBIAL CONSORTIA”; and U.S. patentapplication Ser. No. 11/099,880, titled “GENERATION OF MATERIALS WITHENHANCED HYDROGEN CONTENT FROM MICROBIAL CONSORTIA INCLUDINGTHERMOTOGA,” of which the entire contents of both applications is hereinincorporated by reference for all purposes.

FIG. 1 shows the conversion starting with a complex hydrocarbonsubstrate 102 being metabolized into smaller hydrocarbon products. Thecomplex hydrocarbon substrate may be oil, coal, coke, kerogen,anthracite, coal tar, bitumen, lignite, peat, carbonaceous shale, andsediments rich in organic matter, among other kinds of carbonaceousmaterial. The substrate may also be organic polymers and polyaromatics.

A “first bite” microorganism metabolizes at least a portion of thesubstrate into smaller organic compounds 104. The metabolic reaction mayinvolve the hydrolytic fermentation of the organic substrate intosmaller-sized organic metabolites. These metabolites may be converted byadditional anaerobic microorganisms to even smaller-sized molecules,including carbon dioxide, hydrogen, ammonia, sulfides, and acetate 108.

In the final stages, methanogens may convert the acetate (or hydrogenand carbon dioxide 106) into methane 110. Methanogen metabolism ofacetate may involve an acetate fermentation process that may berepresented by the chemical reaction formula:CH₃COO⁻+H⁺→CH₄+CO₂The methanogen metabolism of hydrogen and carbon dioxide may involve acarbonate reduction process that may be represented by the chemicalformula:CO₂+4H₂→CH₄+2H₂OA single species of methanogen in the consortium may be able to performboth processes, depending on the concentration of acetate, hydrogen andcarbon dioxide in the reaction environment. Alternatively, multiplespecies of methanogens may be present in the consortium, some doingacetate fermentation and some doing carbonate reduction.

In embodiments of the present invention, the “first bite” of thehydrocarbon substrate and the metabolism of the smaller hydrocarbonproduct are catalyzed by a single microorganism. T. phaeum has beenidentified as taking a complex hydrocarbon substrate such as crude oiland metabolizing at least a portion of it into acetate. A secondconsortium member, a methanogen such as Methanosarcina thermophila, maymetabolize the acetate into methane. Thus, a consortium with just twospecies of microorganisms, one of which is T. phaeum, may be used tometabolize a complex hydrocarbon substrate into methane.

Referring now to FIG. 2A, steps in methods of isolating T. phaeum andpreparing a consortium that includes the microorganism according toembodiments of the invention is described. The method includesextracting samples from a formation site 202 that contain a nativeconsortium of microorganisms. The samples may be taken from solidsubstrate and/or formation water at the site. When consortiummicroorganisms include obligate anaerobes, additional steps may be takento isolate the samples in a low oxygen environment. These steps mayinclude sealing the sample in a container that has been purged of airand filled with an atmosphere of nitrogen, helium, argon, etc. Thecontainers may remained sealed until being opened in an anaerobic glovebox for further processing and study.

The formation site samples may be transported to a preparation site,such as a laboratory, so the T. phaeum in the native consortium can beisolated 204. This may include filtering samples of native formationwater to concentrate the microorganisms in the sample. For example, asample of native formation water may be centrifuged through a filterhaving pores that are small enough to trap most microorganism consortiummembers on the filter. A portion of the filtered formation water may beadded back to the filtrate to create a concentrated suspension of thenative consortium.

The samples may be autoclaved to kill or inactivate nativemicroorganisms other than T. phaeum. For example, autoclaving a sampleof formation water at 120° C. for about 20 minutes has been shown toinactivate methanogens in a T. phaeum consortium. The autoclavingprocess may include two, three, or more autoclaving cycles where thesample is repeatedly raised to the peak autoclaving temperature beforebeing lowered back down to room temperature. The T. phaeum itselfsurvives the autoclaving to give an autoclaved sample product ofisolated T. phaeum.

The consortium that includes the T. phaeum isolate may be combined withone or more additional microorganic consortia 206 to create the isolatedmicrobial consortium that can metabolize complex hydrocarbon substratesto materials with enhanced hydrogen content, such as methane andhydrogen. These additional microorganisms may include Thermotogas likeThermotoga hypogea. Other microorganisms that may be included in theisolate may be from genera including Gelria, Clostridia, Moorella,Thermoacetogenium, Pseudomonas, and/or Methanobacter, among others.

Amendments may also be added to the isolated consortium that stimulatethe metabolism of the hydrocarbon substrates. The amendments may includethe addition of minerals, metals, and/or vitamins (“MMV”) to the isolate208. Examples of mineral amendments may include the addition ofchloride, ammonium, phosphate, sodium, magnesium, potassium, and/orcalcium to the isolate, among other kinds of minerals. Metal amendmentsmay include the addition of manganese, iron, cobalt, zinc, copper,nickel, selenate, tungstenate, and/or molybdate to the isolate, amongother kinds of metals. Vitamin amendments may include the addition ofpyridoxine, thiamine, riboflavin, calcium pantothenate, thioctic acid,p-aminobenzoic acid, nicotinic acid, vitamin B12,2-mercaptoehanesulfonic acid, biotin, and/or folic acid, among othervitamins. The addition of these amendments may involve adding mineralsalts, metal salts, and vitamins directly to the isolate, or firstpreparing a solution of the salts and vitamins that then gets added tothe isolate. The concentration of the MMV amendment may depend on theconcentration and composition of an isolated consortium. Examples ofconcentration ranges for amendment components may include about 1 mg/Lto about 500 mg/L for mineral amendment; about 10 μg/L to about 2000μg/L for a metal amendment; and about 1 μg/L to about 100 μg/L for avitamin amendment.

Other amendments may be made to the isolated consortium 210. Forexample, yeast extract may be added to the isolates, and the pH and/ortemperature of the isolates environment may be adjusted.

FIG. 2B shows another flowchart that includes steps in a method ofisolating T. phaeum and making a consortium according to embodiments ofthe invention. The method includes extracting formation water 220 thatincludes T. phaeum and other microorganism species. The formation wateris heated 222 to a temperature that neutralizes (e.g., kills)microorganisms in the water other than T. phaeum. The heating 222 maycause the T. phaeum to form spores that can survive larger variations inenvironmental conditions than active T. phaeum.

Heating the formation water 222 may create an isolate or concentrate ofT. phaeum by neutralizing the other microorganisms present. Additionalisolation steps may also be done to further concentrate and/or purifythe T. phaeum, such as filtering the T. phaeum from other components inthe water. The T. phaeum may then be cultivated 224 to increase theamount of microorganism that gets introduced to the hydrocarbonformation. Cultivation may include germinating T. phaeum spores intoactive microorganisms that can grow in a nutrient rich environment thatstimulates population growth. The environment may also includehydrocarbon substrates that are taken from the formation where the T.phaeum will be introduced. Cultivating the T. phaeum on the samesubstrate may bias growth towards those the members of the T. phaeumpopulation that are most efficient at utilizing the substrate as anutrient source.

The cultivated T. phaeum may be forced into a dormant phase (i.e.,spores) before being stored and/or transported to an introduction site.Spore formation may be accomplished by heating the T. phaeum and/orremoving at least some of the water from the micoorganism's surroundingenvironment. The T. phaeum spores can withstand larger variations inenvironmental conditions (e.g., temperature, hydration, nutrientconcentrations, pH, salinity, etc.) than the active microorganisms,making them easier to store and transport.

A consortium that includes the cultivated T. phaeum may be combined withadditional consortia of microorganisms 226 to form a consortium ofmicroorganisms that includes T. phaeum. The additional consortia mayinclude one or more methanogenic microorganism species (e.g.,Methanosarcina thermophila and Methanosaeta thermophila, etc.).

The combination of the T. phaeum consortium with additional consortiamay take place before or after the T. phaeum is stored and transportedto the formation site where the consortium is introduced 228. The T.phaeum consortium may also be combined with the additional consortia insitu in the hydrocarbon formation. For example, a T. phaeum consortiumand one or more additional consortia may be introduced to the formationin different injection steps and afterwards combine into the finalmicroorganic consortium.

The T. phaeum may be introduced to the hydrocarbon formation 228 asactive microorganisms or as spores that can germinate in situ in theformation environment. The introduction may also include a nutrientamendment that depends on whether the active microorganisms or thespores are being introduced into the hydrocarbon formation. Thetemperature, pH, salinity, and other environmental parameters of the T.phaeum consortium may also depend on whether the T. phaeum is introducedas active microorganisms or spores.

Referring now to FIG. 3, steps in a method of in situ stimulation ofbiological activity of T. phaeum in a geologic formation according toembodiments of the invention are shown. The method may include detectingthe T. phaeum in the formation 302. Detection may be done by extractinga sample from the formation and bringing it to a lab to test for agenetic match, or conducting tests at the formation site. The method mayalso include measuring environmental characteristics in the formation304. These may include temperature, pH, Eh, salinity, atmosphericcomposition, mineralogy, alkalinity, and concentrations of nutrients,vitamins, inorganic elements, terminal electron acceptors, andsubstances that are known to be toxic to T. phaeum, among othercharacteristics of the formation environment.

The method may also include the determination of the composition ofcarbonaceous material the T. phaeum may use as a substrate. Thisdetermination may include the breakdown of major hydrocarbonconstituents of an oil, coal, etc. It may also include an elementalanalysis of the amount of carbon, oxygen, hydrogen, nitrogen,phosphorous, sulfur, metals, etc., present in the material.

The data obtained from the measurements of the environmentalcharacteristics of the formation 304 and composition of the carbonaceousmaterial 306 provides information about the types of amendments that aremost likely to stimulate in situ biological activity of the T. phaeum inthe formation. The biological activity may include increased populationgrowth of T. phaeum relative to other microorganisms in the nativemicroorganism consortium of the formation. It may also includestimulating the rate of metabolic activity of the T. phaeum in theformation.

An amendment with a high probability of stimulating biological activitymay be added to the formation 308. These amendments may include adding aT. phaeum growth stimulant, such as a pyruvate (e.g., sodium pyruvate),to the formation. Amendments may also include adding a compound that isa known nutrient for T. phaeum. These may include alkyl alcohols such asmethanol, ethanol, n-propanol, n-butanol, etc. They may also includediols, such as 2,3,-butanediol, as well as other hydrocarbons such asethanolamine, 3,4,5-trimethoxybenzoate, syringate, vanillate, glyine,cysteine, and formate. They may further include hydrogen and carbondioxide. Amendments may also include the addition of more water to theformation environment to lower the salinity or to spread the in situ T.phaeum over a larger region of the carbonaceous material. They may alsoinclude localized increases in the temperature of the formationenvironment.

The method may also include measuring the rate of biogenic gasproduction 310 after the amendment is added to the formation. Thesemeasurements may involve measuring changes in total gas pressure in theformation, or changes in the partial pressure of a particular gas, suchas methane, hydrogen, carbon monoxide, carbon dioxide, etc. An increasein the presence of a desired product, such as hydrogen and/or methanemay indicate that the degree to which the amendment is stimulatingbiological activity of the T. phaeum. These data may be used todetermine if additional amendments should be made to the formation tofurther stimulate the T. phaeum.

DEFINITIONS

The consortium of microorganisms and methods of using them to makebiogenic materials with enhanced carbon content described here straddlethe arts of microbiology, molecular biology, organic chemistry, and oiland gas recovery, among others. Consequently, some of the terms usedhere may have slightly different meanings in the different arts.Therefore some definitions are provided to clarify the meaning of someterms used to describe the present invention.

“Microorganism,” as used here, includes bacteria, archaea, fungi,yeasts, and molds. Sometimes a microorganism can have definingcharacteristics from more than one class, such as a combination ofbacteria and fungi. The term microorganism as used here also encompassesthese hybrid classes of organisms.

“Anaerobic microorganisms,” as used here, refers to microorganisms thatcan live and grow in an atmosphere having less free oxygen thantropospheric air (i.e., less than about 18%, by mol., of free oxygen).Anaerobic microorganisms include organisms that can function inatmospheres where the free oxygen concentration is less than about 10%by mol., or less than about 5% by mol., or less than about 2% by mol.,or less than about 0.5% by mol.

“Facultative anaerobes,” as used here, refer to microorganisms that canmetabolize or grow in environments with either high or lowconcentrations of free oxygen.

“Methanogen,” as used here, refers to obligate and facultative anaerobicmicroorganisms that produce methane from a metabolic process. Twometabolic pathways commonly seen in methanogens are acetatefermentation, where acetate and a hydrogen ion are metabolized intomethane and carbon dioxide, and carbonate reduction, where carbondioxide and molecular hydrogen are metabolized into methane and water.Classes of methanogens include Methanobacteriales, Methanomicrobacteria,Methanopyrales, Methanococcales, and Mathanosaeta (e.g., Mathanosaetathermophila), among others. Specific examples of methanogens includeThermotoga hypogea, Thermotoga lettingae, Thermotoga subterranean,Thermotoga elfii, Thermotoga martima, Thermotoga neapolitana, Thermotogathernarum, and Thermotoga petrophila, Methanobacterthermoautotorophicus, Methanobacter wolfeii. Methanogens may alsoproduce methane through metabolic conversion of alcohols (e.g.,methanol), amines (e.g., methylamines), thiols (e.g., methanethiol),and/or sulfides (e.g., dimethyl sulfide). Examples of these methanogensinclude methanogens from the genera Methanosarcina (e.g., Methanosarcinabarkeri, Methanosarcina thermophila, Methanosarcina siciliae,Methanosarcina acidovorans, Methanosarcina mazeii, Methanosarcinafrisius); Methanolobus (e.g., Methanolobus bombavensis, Methanolobustindarius, Methanolobus vulcani, Methanolobus taylorii, Methanolobusoregonensis); Methanohalophilus (e.g., Methanohalophilus mahii,Methanohalophilus euhalobius); Methanococcoides (e.g., Methanococcoidesmethylutens, Methanococcoides burtonii); and/or Methanosalsus (e.g.,Methanosalsus zhilinaeae). They may also be methanogens from the genusMethanosphaera (e.g., Methanosphaera stadtmanae and Methanosphaeracuniculi, which are shown to metabolize methanol to methane). They mayfurther be methanogens from the genus Methanomethylovorans (e.g.,Methanomethylovorans hollandica, which is shown to metabolize methanol,dimethyl sulfide, methanethiol, monomethylamine, dimethylamine, andtrimethylamine into methane).

“Materials with enhanced hydrogen content,” as used here, means organicproducts that have a higher ratio of C—H to C—C bonds than the organiccompounds from which they were derived. These materials will also have ahigher mol. % hydrogen than found in the compounds from which they werederived. For example, acetic acid has the chemical formula CH₃COOH,representing 2 carbon atoms, 2 oxygen atoms, and 4 hydrogen atoms, togive a total of 8 atoms. Since 4 of the 8 atoms are hydrogen, the mol. %of hydrogen atoms in acetic acid is: (4 Hydrogen Atoms)/(8 TotalAtoms)=0.5, or 50%, by mol. (or on a molar basis). Methane has thechemical formula CH₄, representing 1 carbon atom and 4 hydrogen atoms,making a total of 5 atoms. The mol. % of hydrogen atoms in methane is (4Hydrogen Atoms)/(5 Total Atoms)=0.8, or 80%, by mol. Thus, theconversion of acetic acid to methane increases the mol. % of hydrogenatoms from 50% to 80%. In the case of molecular hydrogen, the mol. % ofhydrogen atoms is 100%.

“Hydrocarbon,” as used here, means molecules that contain carbon andhydrogen atoms. Optionally, the molecules may also have nitrogen,sulfur, phosphorous, and/or oxygen atoms, among other atomic species.

“Complex Hydrocarbon Substrates,” as used here, include compounds foundin geologic deposits of carbonaceous material. These may include coal,oil, kerogen, peat, lignite, oil shale, tar sands, bitumen, and tar.

EXPERIMENTAL

During a course of experiments to characterize metabolic activity of anative microorganism consortium, a surprising result was observed. FIG.4A charts the robust production of acetate that was measured fromseveral control samples of native formation water and oil that wasautoclaved to kill the microorganisms. FIG. 4B shows that non-autoclavedoil and water incubations did not generate acetate concentrations ashigh as the autoclaved incubations. At first, the presumption was thatacetic acid present in the oil was escaping into the formation water ofthe sample bottles. But testing revealed almost no acetate was presentin the samples. Thus, an abiotic source for the observed acetateproduction seemed unlikely.

Additional examination of the autoclaved formation water samples wasdone, including filtration of the formation water and examination of thefiltered cells with phase-contrast microscopy. Rod-shaped microorganismswere observed in the autoclaved formation water, suggesting at least onespecies was able to survive the autoclaving conditions.

But apparently not all the species in the native consortium survivedautoclaving. Methanogenesis was observed in the non-autoclaved samplesand produced much less acetate than the autoclaved samples (see FIG. 5).This suggested that the methane producing microorganisms were acetateutilizing methanogens that metabolized the acetate into methane. It alsosuggested that autoclaving was effective at killing the methanogens, butnot the acetate generating species.

A Terminal Restriction Fragment Length Polymorphism (TRFLP) analysis ofthe 16S rDNA was run to identify the species that survived theautoclaving and produced the acetate. The TRFLP analyses of theautoclaved samples that produced acetate showed only one significantband. Sequencing of this 16S rDNA band revealed that it was the 16S rDNAgene of Thermacetogenium phaeum (T. phaeum). This suggested T. phaeumwas the sole active microbial species in the autoclaved sample.

This conclusion is somewhat surprising because it suggests a singlespecies can metabolize a complex hydrocarbon substrate to acetate. It'salso one of the few instances where a “first bite” anaerobicmicroorganism that is responsible for the initial metabolism of acomplex hydrocarbon substrate (in this case crude oil) undermethanogenic conditions (e.g., in the absence of electron acceptorsincluding nitrate and sulfate) has been positively identified. Theimplication of this discovery is that a consortium of just two speciesof microorganisms can be constructed that can metabolize complexhydrocarbon substrates into methane. If only two species are necessaryto do this, amendments can be developed to maximize the metabolicactivity of the target species(s) without as much concern about theircollateral effects on other species. Furthermore, since the role of themethanogen can be played by a variety of species, a single methanogenspecies (or group of species) can be selected that has the highestproductivity when paired with T. phaeum.

Additional details of the experiments that lead to the discovery of T.phaeum as a first bite anaerobic microorganism are now described.

Testing Procedures Used with Formation Water Samples

Microbial Activity Experiments: Triplicate experimental incubations wereprepared by adding ten ml of produced water collected from elevenoil-water separators and from a producing well (Nine Mile 4-6 wellhead)to 30 ml serum bottles while working in an anaerobic glove bag. 0.1 mlof oil from the respective sampling locations was added to theincubations. The headspace of the incubations was replaced with N₂/CO₂(95/5).

Nutrient supplemented incubations received solutions of minerals,metals, vitamins, and yeast extract (“MMVYE”) according to Table 1:

TABLE 1 Composition of the MMVYE Nutritional Supplement AmendmentConcentration Mineral Salts Mineral Salt Concentration in mg/L Chloride443 Ammonium 111 Phosphate 23.5 Sodium 103 Magnesium 9.9 Potassium 17.3Calcium 3.6 Metals Metal Concentrations in μg/L Manganese 1600 Iron 570Cobalt 250 Zinc 230 Copper 40 Nickel 20 Selenate 80 Tungsten 70Molybdate 66 Vitamins Vitamin Concentrations in μg/L Pyridoxine HCL 40Thiamine 20 Riboflavin 20 Calcium pantothenate 20 Thioctic acid 20p-aminobenzoic acid 20 Nicotinic acid 20 Vitamin B12 202-mercaptoehanesulfonic acid 8 Biotin 8 Folic acid 8

Autoclaved control samples of formation water and oil were produced byautoclaving on three successive cycles of raised temperature andpressure. For each cycle the formation water sample was heated to 120°C. for 20 minutes under a pressure of 15 psi.

Methane, carbon dioxide, and hydrogen were quantified in the headspaceof the incubations by gas chromatography with flame ionization detection(“GC-FID”). Organic acids in the fluid samples post-incubation were alsoquantified with GC-FID.

Quantification of Acetate in Oils: Five gram samples of several oils(Nine Mile 4-6 well head, Nine Mile 10-7, Monument Butte 12-35, Ashley12-11, and Gilsonite 6-32) were added to serum bottles while workinginside an anaerobic glove bag. The headspace was then exchanged withhelium. The samples were brought to 60° C., and 10 ml of anoxic HPLCwater was injected into the samples. The samples were shaken vigorouslywith a vortex placed inside the 60° C. incubator for thirty minutes. Anaqueous sample was then obtained and analyzed for organic acids by gaschromatography.

Microscopic examination of microorganisms in the samples was done byrecovering 0.4 ml of liquid from experimental bottles MBP-01 NE 100(H₂+Min+Met+Vit+YE) and MBP-01 NE 092 (Sterile control) and centrifugingthem through a sterile 0.2 um filter at 5000 rpm. 40 ul of supernatantwas added back to the filter to suspend the cells. 20 μl of theconcentrated cell suspensions was used for direct microscopicexamination for microbial cells via phase-contrast microscopy. Theremaining 20 μl was used to prepare a gram stain for microscopicexamination via light microscopy.

TRFLP analysis of the samples was done by isolating a DNA fraction frommicroorganism cultures derived from each sample. Approximately 50 ng ofconsortium DNA was used in PCR reactions containing primers 519F(sequence 5′-CAGCMGCCGCGGTAATWC-3′; SEQ ID NO:1) and infrared-labeled(IR)1406R (sequence 5′-ACGGGCGGTGWGTRCAA-3′; SEQ ID NO:2). The 891 basepair product was gel purified and digested with the restriction enzymeHha I. Digestion products were desalted, concentrated to 10 μl in aspeedvac, and 4 μl was electrophoresed on an 8% polyacrylamide gel usingthe Li-Cor 4300 genetic analyzer. After PCR amplification the lightexposure was minimized to avoid photobleaching the infrared label. After30 minutes of electrophoresis predominant IR labeled bands were excisedfrom the gel using the Li-Cor Odyssey and purified with the Qiaex II gelpurification kit (Qiagen). An 18 base double-stranded linker (SL1 andSL1 complement) was ligated to the non-labeled end of theoligonucleotide, and DNA fragments were re-amplified using standard PCRtechniques with primers SL1 and 1406R. PCR products were gel purified,and cloned using the Invitrogen TOPO TA cloning kit. Clones weresequenced using an IR-labeled 1492 primer and the SequiTherm EXCEL™ IIDNA Sequencing Kit (Epicentre).

Experimental Results

FIG. 4A plots the accumulation of acetic acid (i.e., acetate) in theformation water oil-water incubations established with samples from theMB 12-35 production well. FIG. 4B plots methane for the sameincubations. Significant accumulation of acetic acid was observed in MB12-35 oil water incubations that were autoclaved. Methane was notproduced in these autoclaved incubations. In contrast to the autoclavedoil-water incubations only slight acetate accumulation was observed inthe unamended incubations, and the acetate concentration decreased inthe nutrient amended incubations (presumably consumed by themethanogens). As FIG. 4B shows, rapid methanogenic activity was detectedin MMVYE amended incubations, with greater than 12% methane accumulationprior to the first headspace sampling at 10 days. Methanogenic activityoccurred at a slower rate in unamended incubations.

The plots in FIGS. 4A-B indicate that autoclaving was effective atkilling methanogens. The accumulation of acetate in the unamendedincubations indicates that acetate production outpaced acetateconsumption. The addition of MMVYE stimulated acetate conversion tomethane relative to the unamended controls suggesting that downstreammetabolism, including methanogens, was nutrient limited.

The source of acetic acid production could not have been diffusion fromthe oil. Water extractable acetate, propionate, butyrate, isobutyrate,isovalerate, isocaproic acid, hexanoic acid, and heptanoic acid werebelow detection in all of the tested oil samples including the MB 12-35oil. Propionic acid was detected at a relatively low concentration (0.28mM) in the Nine Mile 10-7 oil sample. This argued for either microbialor abiotic generation of acetate in the autoclaved controls.

Formation water in both the MBP-01 MB 12-35 sterile and MBP-01 MB 12-35hydrogen+Minerals+Metals+Vitamins+YE samples were turbid in appearanceand contained numerous microbial cells visualized with the microscope.Significantly higher numbers of microorganisms were observed in thenon-autoclaved sample. This suggested that the sterile controls were notsterile at the time the liquid samples were collected for microscopicexamination. The finding that acetate but not methane is produced in thesterile controls argues that cross contamination from live to sterileincubations during sampling (via sterile syringes and needles) is notthe source of microorganisms in the sterile controls. Crosscontamination would also have transferred methanogens resulting inmethanogenesis. These results argue for a microbial source of acetateproduction in these autoclaved incubations although more definitiveanalyses were conducted for confirmation.

Experimental bottles, including MBP01, NE093 and MBP04NE011, all ofwhich are autoclaved incubations that generated significant acetate,were sampled and analyzed to identify the identity of microorganismswithin the aqueous phase. FIG. 5 shows the TRFLP banding patterns forthe DNA obtained from several MBP-01 incubations.

Thermacetogenium phaeum was the only microorganism detected in theMB12-35 autoclaved incubations analyzed from independent experiments. Incontrast, four different microorganisms, including Thermotoga hypogeawere present in live (non-autoclaved) incubations prepared with theMB12-35 production fluid and oil. These results conclusively indicatethat Thermacetogenium phaeum (or T. phaeum spores) survived three cyclesof autoclaving and that Thermacetogenium phaeum is capable of producingacetate from hydrocarbons contained within the samples.

Acetate production was determined in several autoclaved Monument Butteincubations to further evaluate the significance of the findings withthe MB 12-35 sample. FIGS. 6A-B plot the concentration of acetate inautoclaved control samples (FIG. 6A) and unamended, unautoclavedincubations (FIG. 6B). Very significant acetate production was observedin three of the autoclaved samples and at least some acetate accumulatedin most of the autoclaved samples. In five of twelve samples, acetateaccumulated to a greater extent and at faster rates in autoclavedcontrols, relative to unamended incubations. Interestingly, includedwithin the group of samples that generated high acetate in sterilecontrols were all three samples that generated methane to asignificantly higher extent (>18.5% headspace methane) in thecorresponding unamended controls.

Based on the these results, Thermacetogenium phaeum was identified asthe first bite species that metabolized the starting complex hydrocarbonsubstrate (crude oil) into acetate. Thermacetogenium phaeum is ahomoacetogenic bacterium that can grow on various substrates, such asmethoxylated aromatics, pyruvate, glycine, cysteine, formate andhydrogen/CO₂ . T. phaeum is a gram-positive, spore-forming, androd-shaped microorganism growing optimally at 58° C. and pH 6.8. It canalso grow on acetate if cocultured with a hydrogen-consumingmethanogenic partner, such as Methanothermobacter thermautotrophicus.

Having described several embodiments, it will be recognized by those ofskill in the art that various modifications, alternative constructions,and equivalents may be used without departing from the spirit of theinvention. Additionally, a number of well known processes and elementshave not been described in order to avoid unnecessarily obscuring thepresent invention. Accordingly, the above description should not betaken as limiting the scope of the invention.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassed.The upper and lower limits of these smaller ranges may independently beincluded or excluded in the range, and each range where either, neitheror both limits are included in the smaller ranges is also encompassedwithin the invention, subject to any specifically excluded limit in thestated range. Where the stated range includes one or both of the limits,ranges excluding either or both of those included limits are alsoincluded.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “a process” includes aplurality of such processes and reference to “the electrode” includesreference to one or more electrodes and equivalents thereof known tothose skilled in the art, and so forth.

Also, the words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, acts, orgroups.

1. A method of isolating Thermacetogenium phaeum from a nativeconsortium of microorganisms, the method comprising: removing formationwater from an underground formation, wherein the formation watercomprises the native consortium that includes the Thermacetogeniumphaeum; and heating the formation water to about 120° C. or more,wherein the Thermacetogenium phaeum survives the heating process.
 2. Themethod of claim 1, wherein the formation water is heated for about 20minutes or more.
 3. The method of claim 1, wherein the formation wateris heated at a pressure of about 15 psi or more.
 4. The method of claim1, wherein the formation water is heated in three or more successiveheating cycles that take the formation water from room temperature tothe heating temperature.
 5. The method of claim 1, wherein the formationwater is heated at 120° C. for 20 minutes at 15 psi in three consecutiveheating cycles.
 6. The method of claim 1, wherein at least some of theThermacetogenium phaeum forms spores during the heating process.
 7. Themethod of claim 1, wherein the formation water is heated in anatmosphere with substantially no free oxygen.
 8. The method of claim 7,wherein the atmosphere is nitrogen and carbon dioxide.